The Crystal Structure of the Y66L Variant of Green Fluorescent Protein Supports a Cyclization-Oxidation-Dehydration Mechanism for Chromophore Maturation
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- 作者：Matthew A. Rosenow ; Holly A. Huffman ; Marlene E. Phail ; and Rebekka M. Wachter
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：April 20, 2004
- 年：2004
- 卷：43
- 期：15
- 页码：4464 - 4472
- 全文大小：261K
- 年卷期：v.43,no.15(April 20, 2004)
- ISSN：1520-4995
文摘
The crystal structure of a colorless variant of green fluorescent protein (GFP) containing theY66L substitution has been determined to 1.5 Å. Crystallographic evidence is presented for the formationof a trapped intermediate on the pathway of chromophore maturation, where the peptide backbone ofresidues 65-67 has condensed to form a five-membered heterocyclic ring. The hydroxyl leaving groupremains attached to the ring as confirmed by high-resolution electrospray mass spectrometry. The 
-carbonof residue 66 exhibits trigonal planar geometry, consistent with ring oxidation by molecular oxygen. Sidechain positions of surrounding residues are not perturbed, in contrast to structural results obtained for theGFPsol-S65G/Y66G variant [Barondeau, D. P., Putnam, C. D., Kassmann, C. J., Tainer, J. A., and Getzoff,E. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 12111-12116]. The data are in accord with a reactionpathway in which dehydration is the last of three chemical steps in GFP chromophore formation. A novelmechanism for chromophore biosynthesis is proposed: when the protein folds, the backbone condensesto form a cyclopentyl tetrahedral intermediate. In the second step, the ring is oxidized by molecular oxygen.In the third and final step, elimination of the hydroxyl leaving group as water is coupled to a protontransfer reaction that may proceed via hydrogen-bonded solvent molecules. Replacement of the aromaticTyr66 with an aliphatic residue appears to have a profound effect on the efficiency of ring dehydration.The proposed mechanism has important implications for understanding the factors that limit the maturationrate of GFP.
-carbonof residue 66 exhibits trigonal planar geometry, consistent with ring oxidation by molecular oxygen. Sidechain positions of surrounding residues are not perturbed, in contrast to structural results obtained for theGFPsol-S65G/Y66G variant [Barondeau, D. P., Putnam, C. D., Kassmann, C. J., Tainer, J. A., and Getzoff,E. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 12111-12116]. The data are in accord with a reactionpathway in which dehydration is the last of three chemical steps in GFP chromophore formation. A novelmechanism for chromophore biosynthesis is proposed: when the protein folds, the backbone condensesto form a cyclopentyl tetrahedral intermediate. In the second step, the ring is oxidized by molecular oxygen.In the third and final step, elimination of the hydroxyl leaving group as water is coupled to a protontransfer reaction that may proceed via hydrogen-bonded solvent molecules. Replacement of the aromaticTyr66 with an aliphatic residue appears to have a profound effect on the efficiency of ring dehydration.The proposed mechanism has important implications for understanding the factors that limit the maturationrate of GFP.